There is a considerable interest by biochemists, clinical chemists and pharmaceutical manufacturers in determining the distribution profiles of N-glycans in biological samples, such as for example therapeutic glycoproteins. Glycosylation profile of therapeutic proteins needs to be monitored during development to ensure proper biological properties and during a production, to ensure consistency of the therapeutic product. N-glycans released from glycoproteins by enzymatic cleavage with PNGase F (Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase, EC 3.5.1.52) are typically labeled at their free-reducing terminus with fluorescent dyes for analysis by methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), carbohydrate gel electrophoresis, mass spectrometry and others. Fluorescent labeling of glycans facilitates their sensitive detection as well as contributes to improved resolution. PNGase F-released N-glycans are most commonly labeled by reductive amination, where the free-reducing end of a glycan is conjugated to the free amino group of a fluorescent dye. Fluorescent labeling of glycans by reductive amination usually requires anhydrous conditions, elevated temperatures and extended incubation times, which may result in a partial degradation of biologically important, labile constituents of N-glycans, for example, sialic acids.
PNGase F releases N-glycans from glycoproteins initially as β-glycosylamines, where the free-reducing end of the released glycan is conjugated with ammonia (see, Tarentino, et al. TIGG 1993, 23, 163-170; Rasmussen J. R. J. Am. Chem. Soc. 1992, 114, 1124-1126; Risley, et al. J. Biol. Chem. 1985, 260, 15488-15494, 1985). The stability of glycosylamines is dependent on the pH and lower pH favors rapid hydrolysis of glycosylamines to glycans with free-reducing ends and ammonia. At elevated pH, glycosylamines are stable and hydrolyze slowly, which allows glycans released as glycosylamines to be labeled with reagents reactive toward the amino groups instead of the free-reducing ends. Derivatization of glycosylamines with a number of amine-reactive reagents has been reported (for derivatization with phenylisothiocyanate, see, Rasmussen, J. R. J. Am. Chem. Soc. 1992, 114, 1124-1126; for derivatization with FMOC-Cl, see, Kamoda, et al. J Proteome Res. 2005, 4(1):146-52; for derivatization with FMOC-Cl and other dyes see Kurihara T. et al. Anal. Chem. 2007, 79(22):8694-8).
Non-fluorescent and fluorescent activated carbamate reagents are useful for derivatization of amino groups and for spectrophotometric and fluorometric detection of amino acids (see, Nimura, et al. Anal. Chem. 1986, 58, 2372-2375; Iwaki, et al. J. Chromatography 1987, 407, 273-279, 1987; Cohen, et al. Analytical Biochemistry 1993, 211, 279-287; and U.S. Pat. No. 5,296,599).
Therefore, there is a need in the art for dyes and methods that are capable of rapid labeling of glycans under mild conditions, without causing the degradation of biologically active labile components, and can provide high sensitivity of detection and high resolution during separation of glycans. Surprisingly, the present invention meets these and other needs.